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Protein
& Peptide Letters
ISSN: 0929-8665
Protein
& Peptide Letters
Volume
17, Number 10, 2010
Contents
Regular Papers
Aspartimide Modified Galanin Analogue Antagonizes Galanin
Action on Insulin Secretion Pp. 1182-1188
J. Ruczynski, Z. Konstanski, M. Cybal, I. Kocic and
P. Rekowski
[Abstract] [Purchase
Article]
Both OB Folds of Single-Stranded DNA-Binding
Protein Are Essential for Its ssDNA Binding Activity in Deinococcus
radiodurans Pp. 1189-1197
X. Hua, C. Wang, Y. Zhao, H. Wang, L. Huang,
G. Xu, M. Li, Y. Wang, B. Tian and Y. Hua
[Abstract] [Purchase
Article]
Characteristic Peptides of Protein Secondary Structural
Motifs Pp. 1198-1206
R.R. Joshi and S. Sekharan
[Abstract] [Purchase
Article]
Prediction of Cyclin Proteins Using Chou’s Pseudo Amino
Acid Composition Pp. 1207-1214
H. Mohabatkar
[Abstract] [Purchase
Article]
A Novel Motif Discovery Algorithm for Identifying Protein
Families Pp. 1215-1222
R. Wei, L. Gao and T. Zhang
[Abstract] [Purchase
Article]
Analogues of Trypsin Inhibitor SFTI-1 with Disulfide
Bridge Substituted by Various Length of Carbonyl Bridges
Pp. 1223-1227
A. Legowska, E. Bulak, A. Jaskiewicz, I. Maluch, M. Sieracki,
M. Wysocka, A. Lesner and K. Rolka
[Abstract] [Purchase
Article]
Overproduction of 15N-Labeled
r-RGD-Hirudin in Pichia pastoris for NMR Studies
Pp. 1228-1233
J. Wang, Y. Zhang, S. Li, X. Liu, X. Yan, H. Song,
M. Yu, L. Dai and W. Mo
[Abstract] [Purchase
Article]
Anti-Oxidative Stress and Beyond: Multiple Functions
of the Protein Glutathionylation Pp. 1234-1244
Y. Hu, T. Wang, X. Liao, G. Du, J. Chen and
J. Xu
[Abstract] [Purchase
Article]
Intein-Mediated Expression and Purification of
an Analog of Glucagon-like Peptide-1 in Escherichia coli
Pp. 1245-1250
C. Ma, M. Gao, W. Liu, J. Zhu, H. Tian, X. Gao
and W. Yao
[Abstract] [Purchase
Article]
Kinetics and Docking Studies of a COX-2 Inhibitor
Isolated from Terminalia bellerica Fruits
Pp. 1251-1257
T.C. Reddy, P. Aparoy, N.K. Babu, K.A. Kumar, S.K. Kalangi
and P. Reddanna
[Abstract] [Purchase
Article]
Serum Adiponectin Levels in Patients with Familial
Mediterranean Fever Pp. 1258-1262
G. Keskin, A. Inal and L. Özisik
[Abstract] [Purchase
Article]
Prediction of Subcellular Location of Apoptosis
Proteins Using Pseudo Amino Acid Composition: An Approach
from Auto Covariance Transformation Pp. 1263-1269
T. Liu, X. Zheng, C. Wang and J. Wang
[Abstract] [Purchase
Article]
Inhibition Kinetics of Flavonoids on Yeast α-Glucosidase
Merged with Docking Simulations Pp. 1270-1279
H. Xu
[Abstract] [Purchase
Article]
Expression, Purification, and Characterization
of a Functional Mutant Recombinant Human Interleukin-2 Pp.
1280-1284
M. Liu, B. Wang, G. Sun, D. Qian, Z. Yan, X.
Song and S. Ding
[Abstract] [Purchase
Article]
HDL Stimulates apoM Secretion Pp. 1285-1289
J. Ahnström, O. Axler and B. Dahlbäck
[Abstract] [Purchase
Article]
Synthesis and Bioactivity Evaluation of Dipeptidyl
Peptidase IV Resistant Glucagon-like Peptide-1 Analogues Pp.
1290-1295
J. Zhou, S. Ni, H. Zhang, H. Qian, Y. Chi, W.
Huang, L. Yu, X. Hu and W. Chen
[Abstract] [Purchase
Article]
Expression, Purification, Crystallization and
Preliminary X-Ray Crystallographic Analysis of the Peptidoglycan
Binding Region of the Ser/Thr Kinase PrkC from Staphylococcus
aureus Pp. 1296-1299
A. Ruggiero, F. Squeglia, V. Izzo, A. Silipo,
L. Vitagliano, A. Molinaro and R. Berisio
[Abstract] [Purchase
Article]
Synthesis and Activity of N-Sulfonylamides of
Tripeptides as Potential Urokinase Inhibitors Pp.
1300-1304
A. Markowska, I. Bruzgo, W. Miltyk and K. Midura-Nowaczek
[Abstract] [Purchase
Article]
Identification of N- and C-Terminal Residues Involved
in MAP Kinase Phosphatase 3 (MKP3) Interdomain Binding and
Auto Inhibition Pp. 1305-1310
John K. Mark, Michael J.W. Johnston and
Mary Alice Hefford
[Abstract] [Purchase
Article]
Abstracts
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Aspartimide Modified Galanin Analogue Antagonizes
Galanin Action on Insulin Secretion
J. Ruczynski, Z. Konstanski, M. Cybal, I. Kocic and
P. Rekowski
Aspartimide (Asi) formation is one of the most serious
side reactions that can occur both during solid phase synthesis
and storage of peptides containing aspartic acid. Although
numerous studies on the mechanism of Asi formation conducted
so far, the problem still remains unresolved and relatively
little is known about the impact of this side reaction on
biological properties of such modified peptides. In the present
work we characterized the effect of Asi formation on biological
properties of galanin(1-15) analogue modified in position
14 with aspartic acid, investigating its action on rat isolated
gastric smooth muscles and glucose-induced insulin secretion
from rat isolated islets of Langerhans. Our results show that
this side process may adversely affect biological properties
of such modified peptides. As we expected, modification of
GAL(1-15)NH2 structure
changed the interaction of GAL(1-15)NH2
with its receptors and consequently yielded peptide which,
in studies on insulin secretion, showed insulinotropic- and
antagonistic activities as compared to Asi-free analogue.
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Both OB Folds of Single-Stranded DNA-Binding
Protein Are Essential for Its ssDNA Binding Activity in Deinococcus
radiodurans
X. Hua, C. Wang, Y. Zhao, H. Wang, L. Huang,
G. Xu, M. Li, Y. Wang, B. Tian and Y. Hua
The single-stranded DNA-binding proteins are crucial in all
kinds of DNA metabolic processes. Deinococcus SSB-like
proteins are homodimers in nature and contain two OB folds
per monomer, in contrast to other bacterial SSB proteins that
are functionally active as homotetramers. We generated four
truncated variants of DraSSB protein, based on its crystal
structure (PDB code: 1SE8). Gel filtration showed that DraSSB,
DSCT (lack C-tail) and DSCC (lack C-tail and C-terminal OB)
were mostly homodimers, and DSN (lack N-terminal OB) and DSNC
(lack N-terminal OB and connector) were mostly monomers. The
gel filtration supported the hypothesis that the N-terminal
domain played a predominant role in dimerisation. Biochemical
characterization was used to determine the role of each OB
fold in DNA binding, by EMSA and FRET. EMSA results suggested
that binding of DraSSB to ssDNA substrate needed both N- and
C-terminal OB-folds, and also their interaction to achieve
optimum DNA binding. DSCT might possess two ssDNA binding
modes compared with DraSSB. The C-terminal tail was not essential
for binding of ssDNA substrates. The C-terminal OB-fold had
the ability to bind to the bubble structure. Furthermore,
the FRET results for DSCT verified the hypothesis that DSCT
showed two different binding modes for ssDNA, similarly to
EcoSSB.
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Characteristic Peptides of Protein Secondary Structural
Motifs
R.R. Joshi and S. Sekharan
Characteristic peptides of the protein segments having common
secondary folds are obtained for the I-sites library using
maximal position specific probability scores. The
secondary structures of these peptides are predicted deploying
two best-known computational methods. These are validated
with significant accuracy against the corresponding motifs.
The characteristic peptides also match with those computed
using a Bayesian modeling approach with Markov Chain Monte
Carlo Simulation. Percentage representation of the characteristic
peptides in the protein structural and functional families
shows some interesting results with potential applications
in protein structural genomics.
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Prediction of Cyclin Proteins Using Chou’s Pseudo Amino
Acid Composition
H. Mohabatkar
There are different types of cyclins, which are active during
the cell cycle and enable cyclin-dependent kinases to phosphorylate
different substrates. Since there is not much similarity between
amino acid sequences of cyclins, predicting these proteins
is an important job. This paper presents a bioinformatics
classifier to predict cyclins based on Chou’s pseudo
amino acid composition. Analysis of the results by StAR, which
is a program for the analysis of ROC curves, showed that accuracy
of the approach was 83.53% (AUC=89.44%). The present work
demonstrates that the method can provide useful information
for predicting cyclins.
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A Novel Motif Discovery Algorithm for Identifying Protein
Families
R. Wei, L. Gao and T. Zhang
Discovering a protein motif is an important research topic
in both bioinformatics and protein sciences. This paper presents
a novel motif discovery algorithm which is capable of finding
a motif set to represent a protein family. The algorithm involves
an abstraction method of important features, a location-sensitive
connection approach to link two features, and a repeated connection
procedure to generate a motif set. The novel algorithm is
applied to discovering motifs in 21 ligase subfamilies. The
results show that the obtained motifs are able to represent
the characteristics of the subfamilies effectively. The proposed
algorithm could become a potential useful tool for protein
family prediction.
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Analogues of Trypsin Inhibitor SFTI-1 with Disulfide
Bridge Substituted by Various Length of Carbonyl Bridges
A. Legowska, E. Bulak, A. Jaskiewicz, I. Maluch, M. Sieracki,
M. Wysocka, A. Lesner and K. Rolka
Series of eight new monocyclic analogues of trypsin inhibitor
SFTI-1 was synthesized by the solid phase method. In these
analogues disulfide bridge Cys3 – Cys11 present in native
inhibitor was replaced by different-sized carbonyl bridges
formed by the amino groups of the side chain of Lys, Orn,
Dab or Dap located in positions 3 and/or 11. All analogues
appeared to be potent trypsin inhibitors. The values of association
equilibrium constants determined with bovine β-trypsin
ranging 108 – 109
M-1 with the highest (3.90
x 109 M-1)
determined for analogue containing Lys and Dap in aforementioned
positions. The obtained results clearly shown that this redox
stable modification is well tolerated in the structure of
proteinase inhibitor. It is worth stressing that the procedure
of the introduction of carbonyl bridge into the peptide structure
is straightforward and therefore beneficial for the design
of new enzyme inhibitors.
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Overproduction of 15N-Labeled
r-RGD-Hirudin in Pichia pastoris for NMR Studies
J. Wang, Y. Zhang, S. Li, X. Liu, X. Yan, H. Song,
M. Yu, L. Dai and W. Mo
The novel recombinant hirudin, r-RGD-hirudin, inhibits
thrombin and platelet aggregation. Here, we reported over-expression
of 15N-labeled r-RGD-hirudin
by Pichia pastoris in minimal medium. After extensive
optimization, the yield of active r-RGD-hirudin reached ~600
mg/L when the yeast cells were cultured in a fermenter. The
purified 15N-labeled r-RGD-hirudin
retained full biological activity and was uniformly labeled.
Heteronuclear NMR of the 15N-labeled
r-RGD-hirudin was performed for the first time, and all signals
in the heteronuclear single quantum coherence (HSQC) spectrum
were successfully assigned.
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Anti-Oxidative Stress and Beyond: Multiple Functions
of the Protein Glutathionylation
Y. Hu, T. Wang, X. Liao, G. Du, J. Chen and
J. Xu
Glutathionylation, covalently attaching glutathione(s)
to cysteine residue(s) of a protein, has attracted great attention
in recent years. The importance of glutathionylation was initially
recognized for its role in protecting proteins from irreversible
oxidation; however, more studies indicate that glutathionylation
is also involved in redox regulation under both normal physiological
conditions and oxidative stresses. Potential mechanisms for
the formation of glutathionylated proteins have been proposed.
Despite the differences among the details of these mechanisms,
glutathionylation is generally induced by intermediates including
glutathione disulfide, protein-sulfenic acids, and thiyl radical.
Taking advantages of proteomics techniques, authors have established
methods to identify glutathionylation utilizing 35S-cysteine-
or biotin-labeled glutathione, or anti-GSH antibodies. Glutathionylation
serves multiple roles in cellular biochemistry, such as modulation
of enzymatic activity, glutathione storage, and dynamic regulation
of protein function. Development of more efficient methods
for glutathionylation identification, systematic investigation
of its roles in the context of cellular biochemistry, the
interaction with other types of protein modification, and
its relevance to some health-threatening diseases will be
the wider focus of studies in protein glutathionylation.
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Intein-Mediated Expression and Purification of
an Analog of Glucagon-like Peptide-1 in Escherichia coli
C. Ma, M. Gao, W. Liu, J. Zhu, H. Tian, X. Gao
and W. Yao
To facilitate expression and purification of an analog
of GLP-1 (mGLP-1), an intein system was employed in this study.
A recombinant fusion protein, CBD-DnaB-mGLP-1, was constructed
and expressed in the form of inclusion body. After refolding,
the intein-mediated self-cleavage was triggered by pH and
temperature shift. By using chitin beads column followed by
single step purification, about 2.58 mg of mGLP-1 with the
purity of up to 98% could be obtained from 1 L medium. Tricine-SDS-PAGE,
RP-HPLC, and ESI-MS were undertaken to determine the purity
and molecular weight of mGLP-1. The glucose-lowering activity
of mGLP-1 was also preliminarily determined.
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Kinetics and Docking Studies of a COX-2 Inhibitor
Isolated from Terminalia bellerica Fruits
T.C. Reddy, P. Aparoy, N.K. Babu, K.A. Kumar, S.K. Kalangi
and P. Reddanna
Triphala is an Ayurvedic herbal formulation consisting
of equal parts of three myrobalans: Terminalia chebula,
Terminalia bellerica and Emblica officinalis.
We recently reported that chebulagic acid (CA) isolated from
Terminalia chebula is a potent COX-2/5-LOX dual inhibitor.
In this study, compounds isolated from Terminalia bellerica
were tested for inhibition against COX and 5-LOX. One of the
fractionated compounds showed potent inhibition against COX
enzymes with no inhibition against 5-LOX. It was identified
as gallic acid (GA) by LC-MS, NMR and IR analyses. We report
here the inhibitory effects of GA, with an IC50
value of 74 nM against COX-2 and 1500 nM for COX-1, showing
~20 fold preference towards COX-2. Further docking studies
revealed that GA binds in the active site of COX-2 at the
non-steroidal anti-inflammatory drug (NSAID) binding site.
The carboxylate moiety of GA interacts with Arg120 and Glu524.
Based on substrate dependent kinetics, GA was found to be
a competitive inhibitor of both COX-1 and COX-2, with more
affinity towards COX-2. Taken together, our studies indicate
that GA is a selective inhibitor of COX-2. Being a small natural
product with selective and reversible inhibition of COX-2,
GA would form a lead molecule for developing potent anti-inflammatory
drug candidates.
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Serum Adiponectin Levels in Patients with Familial
Mediterranean Fever
G. Keskin, A. Inal and L. Özisik
Familial mediterranean fever (FMF) is a systemic disorder
characterized by recurrent attacks of fever and polyserositis.
In FMF, several pro-inflammatory cytokines, such as IL-6,
have been found to be elevated during the attacks. In recent
years, it is shown that some proteins originated from adipose
tissue play important role in inflammatory process. One of
them, adiponectin decreases the expression of adhesion molecules
and inhibits the attachment of active macrophages to the endothelial
surface, so that it acts antiinflammatory effect. In this
study, we analyzed the possible role of serum adiponectin
in the pathogenesis of FMF. Thirty five patients with FMF
and 13 healthy controls (5 female,8 male; mean age 22.3 ±
4.2 years) were enrolled in this study. Fifteen patients were
in active stage (6 female, 9 male, mean age; 22.4 ±
4.1 years, mean disease duration 6.1±2.3 years) and
20 patients were in inactive stage (6 female,14 male, mean
age;22.6 ±4.2 years, mean disease duration; 5.7 ±
1.6 years). Serum adiponectin and IL-6 levels were determined
by ELISA. The mean serum adiponectin levels were 5.3 ±1.6
ng/ml in healthy controls, 55.3 ± 21.8 ng/ml in active
FMF patients and 17.1 ± 4.7 ng/ml in inactive FMF patients.
The mean serum IL-6 levels were 1.9 ± 0.4 ng/ml in
healthy controls, 4.7 ± 1.1 ng/ml in active FMF patients
and 2.9 ± 1.3 ng/ml in inactive FMF patients. Serum
adiponectin levels in patients with FMF were significantly
higher than in healthy controls (p<0.001). Serum adiponectin
levels were significantly high both in active FMF patients
and in inactive FMF patients compared with healthy control
(p<0.001, p<0.001 respectively). Serum IL-6 levels were
significantly higher both in patients with active and inactive
disease as compared with healthy controls (p<0.01 and p<0.05
respectively). In FMF, serum adiponectin levels were correlated
with high levels of serum IL-6 in the active and inactive
patients. Serum adiponectin and IL-6 levels were high during
both active and inactive stages in patients with FMF.
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Prediction of Subcellular Location of Apoptosis
Proteins Using Pseudo Amino Acid Composition: An Approach
from Auto Covariance Transformation
T. Liu, X. Zheng, C. Wang and J. Wang
Knowledge of apoptosis proteins plays an important role
in understanding the mechanism of programmed cell death. Thus,
annotating the function of apoptosis proteins is of significant
value. Since the function of apoptosis proteins correlates
with their subcellular location, the information about their
subcellular location can be very useful in understanding their
role in the process of programmed cell death. In the present
study, we propose a novel sequence representation that incorporates
the evolution information represented in the position-specific
score matrices by the auto covariance transformation. Then
the support vector machine classifier is adopted to predict
subcellular location of apoptosis proteins. To verify the
performance of this method, jackknife cross-validation tests
are performed on three widely used benchmark datasets and
results show that our approach achieves relatively high prediction
accuracies over some classical methods.
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Inhibition Kinetics of Flavonoids on Yeast α-Glucosidase
Merged with Docking Simulations
H. Xu
Flavonoids, also called vitamin P, are widely distributed
in plants fulfilling many functions. Yeast α-glucosidase
(YAGH; EC 3.2.1.20), as extensively used target protein for
screening bioactive compounds from medicine plants, was selected
to explore the possible mechanisms of multiple biological
function of flavonoids. The results in this study indicated
that flavonoids, as mixed-type inhibitors, quenched the intrinsic
fluorescence of YAGH by a mixed fluorescence quenching mechanism.
The interaction information between flavonoids and YAGH was
analyzed using a flexible docking method (AutoDock) and showed
that 3’, 4’ dihydroxyl groups of B ring and 3-OH
of C ring played a more important role in the inhibition activity
than other hydroxyl groups, because the 3’, 4’
dihydroxyl groups of B ring directly interacted with the active-site
residues of YAGH to inhibit enzyme activity and 3-OH of C
ring seemed to be necessary to maintain the proper binding
orientation of flavonoid molecules, thereby making the hydroxyl
groups of B ring interact with active-site resi-dues tightly
in the hydrophobic pocket of YAGH. The results supply a basis
for understanding the mechanisms of multiple biological functions
of flavonoids.
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Expression, Purification, and Characterization
of a Functional Mutant Recombinant Human Interleukin-2
M. Liu, B. Wang, G. Sun, D. Qian, Z. Yan, X.
Song and S. Ding
In the current study, a mutant recombinant human interleukin-2
(MhIL-2) was generated using site-directed mutagenesis. The
bacteria transformed with plasmid pET15b-MhIL-2 were cultured
in LB medium containing 0.6mM IPTG for 8 hours at 27°C.
Approximately 90% of His-MhIL-2 was efficiently expressed
in soluble form. Purification efficiency was optimized using
a number of strategies, including nickel ion chelating chromatography,
desalting chromatography, thrombin cleavage and Superdex 75
gel filtration chromatography. The final product had >95%
purity. PBMCs, CD4+ and CD8+ T cell proliferation assays revealed
that one such mutant has identical functional property to
the wild-type hIL-2. In summary, we generated a mutant hIL-2
that is functionally identical to wild-type hIL-2.
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HDL Stimulates apoM Secretion
J. Ahnström, O. Axler and B. Dahlbäck
Apolipoprotein M (apoM) in human plasma is mainly associated
with HDL. A retained signal peptide anchors apoM to the lipoproteins.
To investigate the role of the signal peptide in the transfer
of apoM from the synthesizing cell to the lipopro-teins, wildtype
apoM cDNA and the Q22A mutant,
introducing a signal peptidase cleavage site, were used to
stably transfect HEK293 cells, which intrinsically do not
express apolipoproteins. When cultured under serum-free conditions,
wildtype apoM was, in contrast to Q22A,
poorly secreted. Addition of serum or purified HDL stimulated
secretion of wildtype apoM, which was recovered in the medium
incorporated in HDL. The liver cell line HepG2, which synthesizes
HDL, was cultured under serum-free conditions and found to
secrete apoM as part of an HDL-like particle. In conclusion,
due to its retained signal peptide, apoM is poorly secreted
unless HDL is either coexpressed or added to the culture medium.
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Synthesis and Bioactivity Evaluation of Dipeptidyl
Peptidase IV Resistant Glucagon-like Peptide-1 Analogues
J. Zhou, S. Ni, H. Zhang, H. Qian, Y. Chi, W.
Huang, L. Yu, X. Hu and W. Chen
Glucagon-like peptide -1 (GLP-1) is an incretin hormone
displaying glucose-dependent stimulation of insulin secretion
and trophic effects on the pancreatic β-cells.
However, GLP-1 is rapidly degraded to GLP-1(9-36) by dipeptidyl
peptidase-IV (DPP-IV), which removes the N-terminal dipeptide
His7-Ala8.
The rapid inactivation of GLP-1 in the blood circulation limits
its clinical application. Hence, we replaced the enzymatic
hydrolyzation position Ala8
with other natural amino acids. The GLP-1 analogues were synthesized
rapidly and efficiently under microwave irradiation, using
Fmoc/tBu orthogonal protection strategy. Studies on blood-glucose-lowering
effect of GLP-1 analogues in vivo were undertaken
using 10-week-old male Kunming mice. The metabolic stability
was tested by incubation with dipeptidyl peptidase-IV (DPP-IV).
Generally, Xaa8-GLP-1 analogues
exhibit resistance to DPP-IV degradation in vitro
and stronger hypoglycemic effect than GLP-1. This may help
to understand the structure-activity relationship of GLP-1
analogues.
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Expression, Purification, Crystallization and
Preliminary X-Ray Crystallographic Analysis of the Peptidoglycan
Binding Region of the Ser/Thr Kinase PrkC from Staphylococcus
aureus
A. Ruggiero, F. Squeglia, V. Izzo, A. Silipo,
L. Vitagliano, A. Molinaro and R. Berisio
PrkC is an important Ser/Thr membrane kinase of Staphylococcus
aureus able to bind peptidoglycans through extra-cellular
domains, denominated as PASTA. Upon peptidoglycan binding,
PrkC is activated and stimulates bacterial growth and revival
from latency. The entire extra-cellular region of PrkC (residues
378-664), containing three predicted PASTA domains and an
extra-domain of unknown function, has been successfully crystallized
using vapor-diffusion methods. The structure has been solved
by Multiwavelength Anomalous Dispersion and refinement is
in progress.
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Synthesis and Activity of N-Sulfonylamides of
Tripeptides as Potential Urokinase Inhibitors
A. Markowska, I. Bruzgo, W. Miltyk and K. Midura-Nowaczek
Twelve peptides of the general X-SO2-D-Ser-Ala-Arg-OH
formula (where X = methyl, phenyl, α-tolyl,
p-tolyl, 4-methylbenzyl, 1-naphtyl, 2-naphtyl, 4-chlorophenyl,
4-bromophenyl, 2-mesityl, 2,4,6-triisopropylphenyl, 4-acet-amidophenyl)
were obtained and tested for their effect on the amidolytic
activities of urokinase, thrombin, trypsin, plas-min, t-PA
and kallikrein. 2,4,6-triisopropylphenyl-SO2-D-Ser-Ala-Arg-OH
was the most selective inhibitor of urokinase and α-tolyl-SO2-D-Ser-Ala-Arg-OH
was the most active inhibitor of uPA with Ki
value 24 μM.
The compounds were tested for their in vitro antitumour
activity in the following human breast cancer cells: standard
MCF-7 and estrogen-independent MDA-MB-231. Four of the synthesized
peptides showed cytotoxic effects against MDA-MB-231 cell
lines in the range from 2.9 to 8.5 μM.
The examined compound did not influence to MCF-7 cancer cells.
The synthesized peptides were nontoxic to pig’s erythrocytes.
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Identification of N- and C-Terminal Residues Involved
in MAP Kinase Phosphatase 3 (MKP3) Interdomain Binding and
Auto Inhibition
John K. Mark, Michael J.W. Johnston and
Mary Alice Hefford
Interdomain binding has been shown to play an important
role in the regulation of MAP kinase phosphatase 3 (MKP3),
a phosphatase involved in control of ERK signalling pathways.
In this study the residues in N- and C-terminal domains responsible
for MKP3 interdomain binding are identified. Peptides from
the N-terminal substrate-binding domain of MKP3 were assessed
for their ability to bind the C-terminal catalytic domain
using surface plasmon resonance. The data indicate that the
residues 77-97 (the Post-KIM peptide) in the MKP3 N-terminal
domain are responsible for its binding to the C-terminal catalytic
domain. Residues in the C-terminal domain that might be important
to interdomain binding were identified using data in the existing
literature. Variants in which these residues had been altered
were examined by circular dichroism and enzymatic assays to
ensure retention of their structure and catalytic properties
before being assessed for their ability to bind the Post-KIM
peptide. The data show that glutamic acid 248, asparagine
267 and, to a lesser extent, arginine 299 are important for
the interaction between the MKP3 C-terminal and the N-terminal
domains. The identified residues map to a region on the surface
of the C-terminal domain that appears complementary to the
N-terminal domain surface defined by the Post-KIM peptide.
This interdomain binding site is distinct from the substrate
interaction sites.
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